The horseshoe crabs Limulus polyphemus, Tachypleus tridentatus, Tachypleus gigas and Carcinoscorpius rotundicauda are phylogenetically primitive marine arthropods which have not evolved significantly over the past 300 million years (1).
The horseshoe crab has an open circulatory system containing blue haemolymph, and the only formed element present in the haemolymph is a cell called the amoebocyte (2).
The use of the Limulus amoebocyte lysate (LAL) as an in vitro test for endotoxin resulted directly from the important observation made by Bang (3). He observed that the horseshoe crab underwent a type of disseminated intravascular coagulation (DIC) when generalized infection occurred with marine gram-negative bacteria. This initial in vivo observation was later extended by the discovery that clotting of Limulus haemolymph could be produced in vitro by addition of either viable gram-negative bacteria or purified endotoxin from the cell wall of gram-negative bacteria (2). It was also discovered by the same researchers (1) that the amoebocyte was the source of all of the factors necessary for haemolymph coagulation.
The current application of the test as an in vitro assay for endotoxin is based on the fact that physical disruption of amoebocytes which have been isolated from the haemolymph by centrifugation yields a suspension (LAL) containing the coagulation components which may then only be activated by bacterial endotoxin.
Application of this principle has made the LAL test the most sensitive method available for the detection of gram-negative bacteria, endotoxin and lipopolysaccharide (LPS). LAL prepared by current purification methods can reliably detect 0.1 ng/ml of purified Escherichia coli standard endotoxin. LAL has been shown to detect either bound (cell-associated) or free endotoxin (4) incorporated in the cell walls of virtually all gram-negative bacteria. However, endotoxin prepared by different extraction procedures, from different species, may vary widely in reactivity (5,6). For these reasons, a reference standard endotoxin (RSE) has been prepared by the U.S. Food and Drug Administration (USFDA) as a means for standardization of LAL and rabbit pyrogenicity tests (United States Pharmacopoiea XX) (7).
Amoebocyte lysate of similar reactivity to endotoxin can be prepared from all 4 species of horseshoe crabs.
The clotting processes in Limulidae
The currently known clotting processes in Limulidae are summarized in Table 1. ##STR1##
Cellular coagulogen: Coagulogen consists of a polypeptide chain with internal disulphide bonds which are important for the stability of the polymerizable form of the molecule (8). In Limulus polyphemus, coagulogen was found to consist of 220 amino acids with half-cystine content of 18 residues (9). No free SH group was detected; glycine appears to be the only N-terminal residue, and serine its C-terminal residue (9). Coagulogen is always converted by a serine protease enzyme. After clotting, the gel protein display a helical structure in electron microscopy (10). In Tachypleus tridentatus, the coagulogen comprises 132-135 amino acids including high levels of basic amino acids, with N-terminal alanine and C-terminal phenylalanine. In Limulus, clot formation seems to involve the cleavage of the single Arg-Lys peptide bond on the coagulogen (11,9). The N-peptides interact among themselves in a non-covalent fashion to form the insoluble clot. In Tachypleus tridentatus, the enzymatic formation of gel involves limited proteolysis of the Arg-Gly and Arg-Thr peptide linkages located in the N-terminal portion of coagulogen, thus releasing peptide (12,13). The C-fragment of Limulus mainly contains glutamic and aspartic acids (14). Whereas Liu et al. (9) detected a C-peptide with 45 amino acids in this species, Nakamura et al. ( 13) and Shishikura et al. (15) claimed that this C-peptide had 28 amino acid residues arranged in a species-specific sequence.
Clotting enzyme: The clotting enzyme is a serine protease enzyme. It exists in two active forms with molecular weights of 78,000 and 40,000, respectively, and a very similar amino acid composition, indicating a monomer-dimer relationship (9). In Tachypleus tridentatus, the unreduced clotting enzyme is described as a glycoprotein with a molecular weight of 42,000 which aggregates to form a protein with a MW of 350,000. The clotting enzyme originates from an inactive pro-clotting enzyme. Pro-clotting enzyme can be activated via two independent pathways (Table 1): by the LPS of gram-negative bacteria, or by the (1-3)-.beta.-D-glucans from the cell walls of certain fungi and algae.
LPS-mediated coagulation: Firstly, it was demonstrated that endotoxin or LPS activates a pro-clotting enzyme of the serine protease type (16,11). Secondly, an additional Factor B or pro-activator was also found to be involved in the LPS-induced coagulation of LAL since it activated the pro-clotting enzyme (17,18). This activation probably involves limited proteolysis, i.e. of an arginyl or lysyl-X bond of the pro-clotting enzyme (18). Finally, this pro-activator was observed to be converted to an active Factor B or activator (i.e. trypsin-type serine protease) by another proteolytic enzyme called protease N (Factor C in T. tridentatus) which seems to be LPS-dependent (18,19). These successive findings indicate that this coagulation process represent a complex enzyme cascade which might also include other unknown factors (Table 1).
Anti-clotting factors: An 80 Kd protein from the amoebocyte membrane specifically binds the endotoxin or LPS (9,20). According to the authors, this receptor protein can recognize and immobilize small quantities of LPS in Limulus blood without massive intravascular coagulation. In addition, an anticoagulant (anti-LPS factor) which inhibits LPS-induced coagulation is present in the amoebocytes from the haemolymph of Tachypleus tridentatus and Limulus polyphemus (21). This anticoagulant inhibits the activation of Factor B, but not its activity. The other mediated coagulation pathway is not affected by the anti-LPS factor (21).
(1-3)-.beta.-D-glucan-mediated coagulation: Both the antitumour agent-(1-3)-.beta.-D-glucan and other antitumour polysaccharides have a potent ability to cause gelation of LAL (22) by activating a factor G which acts on pro-clotting enzyme (23).
Different methods for detecting endotoxin by LAL
By far the most frequently used method is the clot test. This method is based on the fact that the end result of the cascade of reactions in LAL when reacting with endotoxin is an opaque gel or clot. The test is performed as follows: 0.1 ml of LAL is mixed with 0.1 ml of test solution in a test tube. The mixture is incubated at 37.degree. C. in a water bath for one hour. The test is recognized as positive when a clot is formed and the clot is stable when the test tube is inverted 180.degree. . Less intense reactions have been described, which manifest as an increase in viscosity, appearing as starch-like granules which adhere to the wall of a test tube, often accompanied by an increased opacity. The subjectivity of interpreting endpoints of less than complete gelation has been a point for criticism of this method of performing the test. This is especially true when clinical specimens are tested because of interference of various substances.
Based on the above-mentioned studies of the reaction mechanism in LAL, other modifications of the LAL test have been published. These include a turbidometric (24), a chromogenic (25), a colorimetric (26), a nephelometric (27), kinetic methods (28-30), different slide methods (31-36), capillary tube methods (38,39) microdilution (40), LAL-bead (41), radioisotopic labelling of coagulogen (42) and an immunoelectrophoretic method which measures the loss of antigenicity of coagulogen when it is cleaved by the LPS-activated enzyme (43). In all these methods, except the immunoelectrophoretic method (43), the test result is read visually or indirectly by means of some sort of equipment.
Specificity of LAL for endotoxin
The specificity of the LAL reaction for endotoxin or LPS has been questioned by several investigators. It has been reported that thrombin, thromboplastin, and certain synthetic polynucleotides all resulted in a positive LAL test (44). Peptidoglycane from gram-positive bacteria (45), exotoxins derived from group A Streptococci (46), several simple polysaccharides, including yeast mannans and bacterial dextrans (47), synthetic dextran derivatives (48), and dithiols (49), cause gelation of LAL.
The LAL test has been used to determine the biological activity of endotoxin-like molecules purified from the membrane material of various organisms. Positive LAL tests were obtained with lipoteichoic acid from Streptococcus faecalis (50), lipoglycanes from different strains of Mycoplasma (51,52), cell wall fractions from Micropolyspora faeni (53) and Chlamydia psittaci (54), and pure preparations of Plasmodium berghei (55), and hot phenol-water extracts of Listeria monocytogenes (56).
Recently, the clinical utility of the LAL test has been disputed. Elin (57) performed a statistical analysis of 17 different studies in humans with LAL and blood and concluded that the clinical utility of the LAL test for the diagnosis of gram-negative septicemia is marginal. Further studies by Tubbs (58) and Galloway et al. (59), respectively, showed that plasma from patients infected with Plasmodium falciparum and plasma from patients infected with Borrelia recurentis reacted positively with the LAL test.
In nearly all of the studies mentioned above, subjective methods have been used to read the LAL test, a positive test being defined as gelation or increased turbidity.
The most difficult challenge for the LAL test has been the detection of endotoxin in blood or plasma. Firstly, the concentration of circulating endotoxin in blood is usually low because of its rapid clearance in the liver. Secondly, plasma contains inhibitors to LAL, endotoxin-inactivating substances, and endotoxin-binding proteins.
Various methods have been used to extract endotoxin from plasma. The best results have been obtained by a combination of dilution and heating of the plasma (43).
Experiments with a rocket-immunoelectrophoretic method to determine the reactivity of LAL with endotoxin (43) has shown a higher degree of accuracy and sensitivity compared with several other methods, and it is suitable for diagnostic purposes.
Immunoelectrophoretic studies of the interaction between LAL and soluble antigens from Pseudomonas aeruginosa and Staphylococcus aureus have shown that LAL is highly reactive with LPS, but can react with other antigens from gram-negative and gram-positive bacteria as well (60).
LAL testing in the pharmaceutical industry
The major user of the LAL test has been the pharmaceutical industry which now performs hundreds of thousands of tests annually on a variety of large and small volume parenteral fluids, biochemicals, and medical devices. The LAL test is now commonly performed for both in-process and final-release testing of many injectable fluids and invasive medical devices produced by the pharmaceutical industry all over the world.
Clinical applications of the LAL test
The clinical applications of the LAL test have been thoroughly discussed by Jorgensen (61). The conclusion which may be drawn from this review of the clinical applications of the LAL test is that a number of potentially successful uses have been described since the introduction of the test in the late 1960s. They have all benefited from the speed, sensitivity, and general specificity of the LAL test for bacterial endotoxin, whether it be bound endotoxin from viable gram-negative bacteria or free endotoxin as the residual evidence of prior or periodic bacterial growth. In all of these applications, the LAL test has shown to be superior in some respects to conventional methods of diagnosing bacterial endotoxins. However, none of these applications have replaced conventional methods so that LAL testing alone is used for diagnosis. Instead, the LAL test is a powerful adjunctive means of determining the presence of gram-negative bacteria in a variety of clinical cases. It has current valid diagnostic uses (meningitis, ocular infections, bacteriuria) and may eventually lead to a better understanding of other pathophysiological consequences of endotoxin, i.e. endotoxemia.